Directed evolution has been widely employed to enhance protein structural stability and activity, expand functionality and alter specificity. We propose a novel approach for generating libraries of variant genes that takes advantage of the evolutionary history of a gene family by generating a combinatorial library incorporating amino acid replacements inferred during episodes of adaptive evolution. We have termed our approach Reconstructing Adaptive Evolutionary Paths (REAP). The utility and feasibility of the REAP approach will be tested on two diverse datasets during the Phase I period. One will consist of the green fluorescent protein-like family, and represents a case with a large body of protein engineering and mutational data is available for comparison. The other dataset will consist of the Lacl/PurR/GaIR bacterial repressers family, and represents an example of a more complex protein that binds both small molecule ligands and DMA. This proposal builds the Foundation's expertise in molecular evolution and on Blue Heron's technology for gene synthesis and library generation. [unreadable] [unreadable] [unreadable]